Macrophages are essential in the early immune response against many pathogens. Besides killing pathogens they can also initiate the adaptive immune response by presenting antigens to T cells. Interferon gamma (IFNy) and tumor necrosis factor (TNF) are essential cytokines for successful clearance of many infectious agents. Activation of macrophages by synergistic effects of these cytokines leads to upregulation of many effector molecules that can kill pathogens or inhibit their growth. To devise strategies to improve the early defense against a variety of pathogens it is important to understand individual genetic variation in the response to pathogens. Our hypothesis is that many genetic differences in disease resistance are due to differences in the macrophage response to pathogens and/or to the effects of IFNy+TNF. Our goal is therefore to identify the genes mediating the variation in the macrophage response to IFNy+TNF and to infection with bioterrorism agents. To do this we will measure the transcriptome of IFNy+TNF-stimulated or infected macrophages from recombinant inbred mice derived from crosses between pathogen susceptible and resistant mice. We will then identify mouse genomic regions that affect transcription levels of individual genes using quantitative trait locus (QTL) analysis. Naive or IFNy+TNF-stimulated macrophages will also be infected with various pathogens and assayed for ability to inhibit the pathogen's growth. Genomic regions affecting the macrophage response will be compared to previously identified genomic regions affecting disease resistance. The identification of a common chromosomal location for both expression QTLs and disease trait QTLs will be used to nominate genes in the disease susceptibility locus. These will be tested in an in vitro killing assay by knocking down the gene using RNAi and measuring the effect on the macrophage's ability to kill the pathogen. We will use Toxoplasma gondii and Francisella tularensis, NIAID category B and A bioterrorism agents that replicate in a parasitophorous vacuole vs the cytoplasm, respectively. We expect to gain a deeper understanding of the molecular basis for genetic variation in the host response to pathogens with a distinct intracellular lifestyle.